Rotaviruses, members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. In the infectious rotavirus particle, the genome is enclosed within a non-enveloped icosahedron capsid composed of three concentric protein layers. The innermost protein layer is a smooth, thin, pseudo T=1 assembly formed from 12 decamers of the core lattice protein VP2. Tethered to the underside of VP2 layer are complexes comprised of the viral RNA-dependent RNA polymerase (RdRP) VP1 and the RNA-capping enzyme VP3. Together, VP1, VP2, VP3, and the dsRNA genome form the core of the virion. The core proteins function together to transcribe the segmented dsRNA genome, producing eleven capped plus-sense +RNAs. The viral RdRP uses the +RNAs as templates for the synthesis of the dsRNA genome. Although the RdRP alone can recognize viral +RNAs, the polymerase is only active when VP2 is present. The VP2-dependent activity of VP1 provides a means by which genome replication (dsRNA synthesis) can be linked with genome packaging (core assembly). [unreadable] [unreadable] An important goal of this project is to define the structure and function of VP1 and to elucidate the mechanism by which VP2 activates VP1. The studies below were performed in the last year to address this goal. [unreadable] [unreadable] (1) The atomic structure of VP1 was determined by X-ray crystallography though a collaboration with Dr. Steve Harrison's group at Harvard. The results show that the rotavirus polymerase is structurally quite similar to that of the reovirus RdRP, both representing compact globular proteins with three distinct domains: (i) an N-terminal protruding domain, (ii) a polymerase domain comprised of fingers, palm, and thumb subdomains, and (iii) a C-terminal bracelet domain. Together, the N- and C-terminal domains of VP1 sandwich most of the polymerase domain, creating a cage structure with the catalytic region located within a largely hollow center. Four tunnels connect the surface of VP1 to the catalytic center. These tunnels allow for (i) entry of nucleotides, (ii) entry of single-stranded (ss) template RNA, (iii) exit of the dsRNA product or (-)RNA template, and (iv) exit of +RNA transcripts. Interestingly, a m7G-cap-binding site is situated near the template entry tunnel. This site may be used by VP1 to maintain a stable interaction with the 5'-end of the plus-strand of dsRNA during transcription.[unreadable] [unreadable] Near the catalytic site of the VP1 is a flexible structural element, called a priming loop, that is predicted to bind the triphosphate of the priming nucleotide during initiation of RNA synthesis. Unlike the priming loop of the reovirus RdRP, the VP1 priming loop is in a retracted conformation, such that it is incapable of supporting initiation. Another unique feature of VP1 is the presence of a plug, formed by the extreme C-terminus, which extends down through the dsRNA/-RNA exit tunnel to a position close to the catalytic site. Analysis of VP1 mutant proteins in cell-free replication assays indicates that the plug has no essential role in RNA synthesis. A possible function of the VP1 plug is to cleave the transient dsRNA hybrid formed at the active site during transcription, allowing the +RNA product and the -RNA template to be directed to separate exit tunnels.[unreadable] [unreadable] (2) Earlier analyses have shown that VP1 specifically recognizes a highly conserved sequence 3'CS (UGUGACC) that is present at the 3'-ends of rotavirus +RNAs. To define the nature of this interaction, VP1 crystals were soaked with relevant RNA oligonucleotides and the complexes generated were analyzed by X-ray diffraction. We found that amino acid residues of the template entry tunnel form base-specific hydrogen bonds with the UGUG portion of the 3'CS. In contrast, amino acids in the tunnel hydrogen bond only with the sugar-phosphate backbone of the ACC nucleotides of the 3'CS. A remarkable observation is that the extensive network of hydrogen bonds formed between VP1 and the 3'CS place the 3'-terminal nucleotide (C1) past the site required to support initiation. Thus, the observation that VP1 alone lacks catalytic activity is not surprising given that its priming nucleotide is not engaged by the retracted priming loop and that its interaction with the 3'CS leaves the terminal C1 nucleotide out-of-register for initiation. The capacity of VP2 to serve as an essential cofactor to the catalytic activity of VP1 implies that interaction between these proteins trigger conformational changes in the VP1 priming loop and in the register of the C1 residue that allow for initiation of dsRNA synthesis. [unreadable] [unreadable] (3) Regions of VP2 involved in VP1 activation were determined by mutagenesis. Sequence analysis of various strains of rotavirus has revealed that VP2 proteins are highly conserved overall, but show marked variation at their N-termini. This region protrudes inwardly from the VP2 shell of the T=1 core and is predicted to be important for VP1-binding. To investigate the functional role of the N-terminus, we generated recombinant VP2 proteins (SA11 strain) containing mutations and assayed them for the capacity to support in vitro dsRNA synthesis. We found that progressive deletion of the VP2 N-terminus resulted in correlative decreases in VP1-mediated dsRNA synthesis, suggesting that the length of the internal protrusion is important to the role of VP2 in VP1 activation. Interestingly, the observation that a VP2 mutant lacking the entire VP2 N-terminal protrusion remained capable of supporting low levels of dsRNA synthesis demonstrated that residues in the VP2 shell domain play an unexpected role in VP1 activation. To evaluate the importance of the N-terminus without introducing deletions into VP2 that might cause structural anomalies, a chimeric protein was engineered in which the SA11 VP2 N-terminus was replaced with that of a highly divergent group C rotavirus (Bristol strain). This chimeric protein showed levels of activity indistinguishable from wild-type SA11 VP2 in our in vitro assay, indicating that VP1 interactions with the N-terminal protrusion are not sequence-specific. [unreadable] [unreadable] Together, the results of these VP2 mutagenesis studies are consistent with the following model of VP1 activation. During the early stages of core assembly, VP1 bound to a +RNA template makes an initial interaction with the internal, N-terminal structure of a VP2 decamer unit. This interaction is dependent upon the length, charge, or structure of the VP2 N-terminus rather than specific residues. Following sequestration of VP1, a second interaction occurs between the RdRP and the shell domain of VP2, which induces conformational changes in VP1 that allow for initiation of dsRNA synthesis. It is not clear whether dsRNA synthesis occurs during or after formation of the T=1 shell. Nonetheless, the requirement for VP2 effectively coordinates RV RNA packaging and genome replication, as well as ensures that viral dsRNAs are not made until cores are available for their protection.